Viruses comprising mutant ion channel protein

ABSTRACT

A method to prepare viruses lacking ion channel activity is provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/834,095, filed Apr. 12, 2001, now U.S. Pat. No. 6,872,395, issuedMar. 29, 2005, which application claims priority under 35 U.S.C. 119(e)from U.S. Provisional Application Ser. No. 60/197,209 filed Apr. 14,2000, which applications are incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with a grant from the Government of the UnitedStates of America (grants AI-29599, AI-42774 and AI-44386 from theNational Institute of Allergy and Infectious Diseases). The Governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

Cell membranes consist of a double layer of lipid molecules in whichvarious proteins are embedded. Because of its hydrophobic interior, thelipid bilayer of cell membranes serves as a barrier to the passage ofmost polar molecules and therefore is crucial to cell viability. Tofacilitate the transport of small water-soluble molecules into or out ofcells or intracellular compartments, such membranes possess carrier andchannel proteins. Ion channels are essential for many cellularfunctions, including the electrical excitability of muscle cells andelectrical signaling in the nervous system (reviewed by Alberts et al.,1994). They are present not only in all animal and plant cells, as wellas microorganisms, but also have been identified in viruses (Ewart etal., 1996; Piller et al., 1996; Pinto et al., 1992; Schubert et al.,1996; Sugrue et al., 1990; Sunstrom et al., 1996), where they arethought to play an important role in the viral life cycle.

The influenza A virus is an enveloped negative-strand virus with eightRNA segments encapsidated with nucleoprotein (NP) (reviewed by Lamb andKrug, 1996). Spanning the viral membrane are three proteins:hemagglutinin (HA), neuraminidase (NA), and M2. The extracellulardomains (ectodomains) of HA and NA are quite variable, while theectodomain domain of M2 is essentially invariant among influenza Aviruses. The life cycle of viruses generally involves attachment to cellsurface receptors, entry into the cell and uncoating of the viralnucleic acid, followed by replication of the viral genes inside thecell. After the synthesis of new copies of viral proteins and genes,these components assemble into progeny virus particles, which then exitthe cell (reviewed by Roizman and Palese, 1996). Different viralproteins play a role in each of these steps. In influenza A viruses, theM2 protein which possesses ion channel activity (Pinto et al., 1992), isthought to function at an early state in the viral life cycle betweenhost cell penetration and uncoating of viral RNA (Martin and Helenius,1991; reviewed by Helenius, 1992; Sugrue et al., 1990). Once virionshave undergone endocytosis, the virion-associated M2 ion channel, ahomotetrameric helix bundle, is believed to permit protons to flow fromthe endosome into the virion interior to disrupt acid-labile M1protein-ribonucleoprotein complex (RNP) interactions, thereby promotingRNP release into the cytoplasm (reviewed by Helenius, 1992). Inaddition, among some influenza strains whose HAs are cleavedintracellularly (e.g., A/fowl plagues/Rostock/34), the M2 ion channel isthought to raise the pH of the trans-Golgi network, preventingconformational changes in the HA due to conditions of low pH in thiscompartment (Hay et al., 1985; Ohuchi et al., 1994; Takeuchi and Lamb,1994).

Evidence that the M2 protein has ion channel activity was obtained byexpressing the protein in oocytes of Xenopus laevis and measuringmembrane currents (Pinto et al., 1992; Wang et al., 1993; Holsinger etal., 1994). Specific changes in the M2 protein transmembrane (TM) domainaltered the kinetics and ion selectivity of the channel, providingstrong evidence that the M2 TM domain constitutes the pore of the ionchannel (Holsinger et al., 1994). In fact, the M2 TM domain itself canfunction as an ion channel (Duff and Ashley, 1992). M2 protein ionchannel activity is thought to be essential in the life cycle ofinfluenza viruses, because amantadine hydrochloride, which blocks M2 ionchannel activity (Hay et al., 1993), inhibits viral replication (Katoand Eggers, 1969; Skehel et al., 1978). However, a requirement for thisactivity in the replication of influenza A viruses has not been directlydemonstrated.

Generally, influenza vaccines have been prepared from live, attenuatedvirus or killed virus which can grow to high titers. Live virus vaccinesactivate all phases of the immune system and stimulate an immuneresponse to each of the protective antigens, which obviates difficultiesin the selective destruction of protective antigens that may occurduring preparation of inactivated vaccines. In addition, the immunityproduced by live virus vaccines is generally more durable, moreeffective, and more cross-reactive than that induced by inactivatedvaccines. Further, live virus vaccines are less costly to produce thaninactivated virus vaccines. However, the mutations in attenuated virusare often ill-defined.

Thus, what is needed is a method to prepare recombinant attenuatedinfluenza virus for vaccines e.g., attenuated viruses having definedmutation(s).

SUMMARY OF THE INVENTION

The invention provides an isolated and purified recombinant viruscomprising a mutant ion channel protein which lacks or has reduced ionchannel activity relative to the activity of a corresponding wild-typeion channel protein. The activity of an ion channel protein may bemeasured by methods well-known to the art, see, e.g., Holsinger et al.(1994). The recombinant viruses of the invention replicate in vitro butare attenuated in vivo. Preferably, the virus is a recombinantorthomyxovirus, e.g., a recombinant influenza virus, or a recombinantlentivirus, e.g., human immunodeficiency virus (HIV). Also preferably,the mutant ion channel protein is a mutant viral ion channel protein. Inone embodiment of the invention, the mutant ion channel proteincomprises at least one amino acid substitution relative to thecorresponding wild-type ion channel protein. The substitution(s) in theion channel protein may be positioned in the ectodomain, the TM domain,or the cytoplasmic domain, or any combination thereof. Preferredsubstitutions are in or near the TM domain of the ion channel protein.For example, for influenza A virus, substitutions may be at residues25-43 of M2, i.e., the TM domain, and preferably are at positions 27,30, 31, 34, 38, and/or 41 of the TM domain of M2. In another embodimentof the invention, the mutant ion channel protein comprises a deletion inat least a portion of the ectodomain, the TM domain, the cytoplasmicdomain, or any combination thereof. Preferably, the deletion is in ornear the TM. In yet another embodiment of the invention, the mutant ionchannel protein is a chimeric protein comprising a portion of an ionchannel protein, e.g., the ectodomain and/or the cytoplasmic domain of aviral ion channel protein, and a heterologous protein, e.g., the TMdomain of a heterologous protein. Also within the scope of the inventionis a recombinant virus comprising a mutant ion channel proteincomprising at least one amino acid substitution, a deletion, aninsertion, a functional portion of a heterologous protein, e.g., aportion which provides a structure such as a TM domain or has a similaractivity as the corresponding portion in the full length heterologousprotein such as catalytic activity, binding to a ligand, or other has adetectable phenotype, or any combination thereof.

As described hereinbelow, recombinant influenza A viruses with defectiveM2 ion channel activity were prepared using a reverse-genetics system(see Example 1 and Neumann et al., 1999). Unexpectedly, all of the M2ion channel mutants replicated as efficiently as the wild-type virus invitro, although their growth was attenuated in mice. Recombinant viruseswere also prepared which comprise a mutant M2 ion channel protein whichis a chimeric protein, e.g., a chimeric protein in which the TM domainof M2 was replaced with the TM domain from HA or NA. These recombinantviruses replicated well in tissue culture, but were highly attenuated inmice. Thus, M2 ion channel activity is not essential for the life cycleof influenza A viruses. Rather, it may serve an auxiliary function thatcould, for example, promote viral replication in vivo. Given that theadministration of E. coli-derived particles, spontaneously formed from afusion protein containing the M2 ectodomain and a portion of thehepatitis B core protein, to mice resulted in 90-100% protection againstlethal virus challenge (Neirynck et al., 1999), and that cold-adaptedlive vaccines are efficacious in humans, the attenuated growth of mutantM2 ion channel viruses in vivo, but not in vitro, indicates that thesemutant viruses may be useful in the development of live influenzavaccines. Thus, the invention further provides a vaccine or immunogeniccomposition comprising the recombinant virus of the invention, and amethod of using the vaccine or immunogenic composition to immunize avertebrate or induce an immune response in a vertebrate, respectively.

Also provided is a method of preparing a recombinant influenza viruscomprising a mutant ion channel protein which lacks or has reducedactivity relative to the corresponding wild-type ion channel protein.The method comprises contacting a host cell with a compositioncomprising a plurality of influenza vectors, including a vectorcomprising a mutant ion channel protein, so as to yield recombinantvirus. For example, for influenza A, the composition comprises: a) atleast two vectors selected from a vector comprising a promoter operablylinked to an influenza virus PA cDNA linked to a transcriptiontermination sequence, a vector comprising a promoter operably linked toan influenza virus PB1 cDNA linked to a transcription terminationsequence, a vector comprising a promoter operably linked to an influenzavirus PB2 cDNA linked to a transcription termination sequence, a vectorcomprising a promoter operably linked to an influenza virus HA cDNAlinked to a transcription termination sequence, a vector comprisingpromoter operably linked to an influenza virus NP cDNA linked to atranscription termination sequence, a vector comprising a promoteroperably linked to an influenza virus NA cDNA linked to a transcriptiontermination sequence, a vector comprising a promoter operably linked toan influenza virus M cDNA linked to a transcription terminationsequence, and a vector comprising a promoter operably linked to aninfluenza virus NS cDNA linked to a transcription termination sequence,wherein the M cDNA comprises mutant ion channel protein DNA; and

b) at least two vectors selected from a vector comprising a promoteroperably linked to a DNA segment encoding influenza virus PA, a vectorcomprising a promoter operably linked to a DNA segment encodinginfluenza virus PB1, a vector comprising a promoter operably linked to aDNA segment encoding influenza virus PB2, a vector comprising a promoteroperably linked to a DNA segment encoding influenza virus NP, a vectorcomprising a promoter operably linked to a DNA segment encodinginfluenza virus HA, a vector comprising a promoter operably linked to aDNA segment encoding influenza virus NA, a vector comprising a promoteroperably linked to a DNA segment encoding influenza virus M1, a vectorcomprising a promoter operably linked to a DNA segment encoding an ionchannel protein, preferably a mutant ion channel protein, and a vectorcomprising a promoter operably linked to a DNA segment encodinginfluenza virus NS2. Preferably, the mutant ion channel protein in thevector of a) or b) is a mutant M2 ion channel protein, e.g., one havingat least one amino acid substitution, deletion, insertion, orheterologous sequence, which lacks or has reduced ion channel activityrelative to the activity of a corresponding wild-type M2 ion channelprotein. The invention further provides a composition such as thatdescribed hereinabove, and a host cell contacted with such a compositione.g., so as to yield infectious virus. Alternatively, the host cell maybe contacted with each vector, or a subset of vectors, sequentially.

The invention also provides a vector encoding a chimeric proteincomprising the ectodomain of an influenza virus ion channel proteinlinked to a heterologous transmembrane protein, preferably linked to acytoplasmic domain, e.g., from an influenza protein which has atransmembrane domain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic diagram of established reverse genetics systems. Inthe RNP transfection method (A), purified NP and polymerase proteins areassembled into RNPs with use of in vitro-synthesized vRNA. Cells aretransfected with RNPs, followed by helper virus infection. In the RNApolymerase I method (B), a plasmid containing the RNA polymerase Ipromoter, a cDNA encoding the vRNA to be rescued, and the RNA polymeraseI terminator is transfected into cells. Intracellular transcription byRNA polymerase I yields synthetic vRNA, which is packaged into progenyvirus particles upon infection with helper virus. With both methods,transfectant viruses (i.e., those containing RNA derived from clonedcDNA), are selected from the helper virus population.

FIG. 2. Schematic diagram of the generation of RNA polymerase Iconstructs. cDNAs derived from influenza virus were amplified by PCR,digested with BsmBI and cloned into the BsmBI sites of the pHH21 vector(E. Hoffmann, Ph.D. thesis, Justus, Liebig-University, Giessen,Germany), which contains the human RNA polymerase I promoter (P) and themouse RNA polymerase I terminator (T). The thymidine nucleotide upstreamof the terminator sequence (*T) represents the 3′ end of the influenzaviral RNA. Influenza A virus sequences are shown in bold face letters.

FIG. 3. Proposed reverse genetics method for generating segmentednegative-sense RNA viruses. Plasmids containing the RNA polymerase Ipromoter a cDNA for each of the eight viral RNA segments, and the RNApolymerase I terminator are transfected into cells together with proteinexpression plasmids. Although infectious viruses can be generated withplasmids expressing PA, PB1, PB2, and NP, expression of all remainingstructural proteins (shown in brackets) increases the efficiency ofvirus production depending on the virus generated.

FIG. 4. Detection of the FLAG epitope in cells infected with atransfectant virus. Antibody staining was used to identify the NA inMDCK cells infected with either PR8-WSN-FL79 (A, D) or A/WSN/33wild-type virus (B, E), or on mock-infected MDCK cells (C, F). Ninehours after infection, cells were fixed with paraformaldehyde, treatedwith Triton X-100 and incubated with either anti-FLAG (A-C) or anti-WSNNA (D-F) monoclonal antibodies. Intensive Golgi staining (red) isapparent in positive samples (A, D, and E).

FIG. 5. Recovery of PA mutants. The PA gene of each virus was amplifiedby RT-PCR with primers that yield a 1226 bp fragment (position 677 to1903 of the mRNA, lanes 1, 3, 5), which was then digested with therestriction enzyme Bsp120I (at position 846 of the mRNA, lanes 4, 7) orPvuII (at position 1284 of the mRNA, lanes 2, 6). The presence ofBsp120I or PvuII sites in the PCR products yielded either 169 bp and1057 bp or 607 bp and 619 bp fragments, respectively. MW=molecularweight markers.

FIG. 6. (A) Primers employed to amplify influenza sequences PB2, PB1, PAand HA and (B) primers employed to amplify influenza sequences NP, NA, Mand NS.

FIG. 7. Schematic diagram of mutant influenza virus M2 proteins andtheir properties. The amino acid sequence of the TM domain (residues 25to 43) is shown in single-letter code in the expanded section of thediagram. The ion channel activity was determined by Holdinger et al.(1994), using a two-electrode voltage-clamp procedure. +, detectable ionchannel activity; −, nondetectable ion channel activity.

FIG. 8. Growth curves of the M2 mutant and wild-type WSN-UdM viruses.MDCK cells were infected with virus at an MOI of 0.001. At the indicatedtimes after infection, the virus titer in the supernatant wasdetermined. The values are means of triplicate experiments. The SD isless than 0.59 for each sample.

FIG. 9. Amantadine sensitivity of the M2 ion channel mutants. The mutantand wild-type WSN-UdM viruses were tested for plaque-forming capacity inMDCK cells in the presence of different concentrations of amantadine.Experiments were performed three times, with the results reported asmeans±SD.

FIG. 10. Schematic diagram of the chimeric M2 mutants. Each mutant wasconstructed by replacing the TM domain of M2 with that of the HA or NA.

FIG. 11. Incorporation of the M2 mutants into influenza virions.Purified viruses were lysed in a sample buffer. Viral proteins weretreated with 2-mercaptoethanol, separated by 15% SDS-PAGE, transferredto a PVDF membrane, and detected with the 14C2 anti-M2 monoclonalantibody (Zebedee and Lamb, 1988) and anti-WSN-NP monoclonal antibody.Molecular masses of the marker proteins are shown on the left.

FIG. 12. Virus-specific antibodies in nasal wash (A), lung wash (B) orsera (C) from vaccinated mice. Mice were intranasally immunized with 50μl of 1.1×10⁵ PFU/ml of M2del29-31 or wild-type WSN-UdM (control)viruses. In the second week after immunization, four mice weresacrificed to obtain samples.

FIG. 13. Body weights of immunized mice which were challenged withwild-type virus two weeks (A), one month (B) or two months (C) afterimmunization.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “isolated and/or purified” refer to in vitropreparation, isolation and/or purification of a vector, plasmid or virusof the invention, so that it is not associated with in vivo substances,or is substantially purified from in vitro substances. An isolated viruspreparation of the invention is generally obtained by in vitro cultureand propagation and is substantially free from other infectious agents.As used herein, “substantially free” means below the level of detectionfor a particular infectious agent using standard detection methods forthat agent. A “recombinant” virus is one which has been manipulated invitro, e.g., using recombinant DNA techniques to introduce changes tothe viral genome.

As used herein, the term “recombinant nucleic acid” or “recombinant DNAsequence or segment” refers to a nucleic acid, e.g., to DNA, that hasbeen derived or isolated from a source, that may be subsequentlychemically altered in vitro, so that its sequence is not naturallyoccurring, or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in the native genome. An exampleof DNA “derived” from a source, would be a DNA sequence that isidentified as a useful fragment, and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.

Orthomyxoviruses

Influenza Virus A

Influenza A viruses possess a genome of eight single-strandednegative-sense viral RNAs (vRNAs) that encode a total of ten proteins.The influenza virus life cycle begins with binding of the HA to sialicacid-containing receptors on the surface of the host cell, followed byreceptor-mediated endocytosis. The low pH in late endosomes triggers aconformational shift in the HA, thereby exposing the N-terminus of theHA2 subunit (the so-called fusion peptide). The fusion peptide initiatesthe fusion of the viral and endosomal membrane, and the matrix protein(M1) and RNP complexes are released into the cytoplasm. RNPs consist ofthe nucleoprotein (NP), which encapsidates vRNA, and the viralpolymerase complex, which is formed by the PA, PB1, and PB2 proteins.RNPs are transported into the nucleus, where transcription andreplication take place. The RNA polymerase complex catalyzes threedifferent reactions: synthesis of an mRNA with a 5′ cap and 3′ polyAstructure, of a full-length complementary RNA (cRNA), and of genomicvRNA using the cDNA as a template. Newly synthesized vRNAs, NP, andpolymerase proteins are then assembled into RNPs, exported from thenucleus, and transported to the plasma membrane, where budding ofprogeny virus particles occurs. The neuraminidase (NA) protein plays acrucial role late in infection by removing sialic acid fromsialyloligosaccharides, thus releasing newly assembled virions from thecell surface and preventing the self aggregation of virus particles.Although virus assembly involves protein-protein and protein-vRNAinteractions, the nature of these interactions is largely unknown.

Although influenza B and C viruses are structurally and functionallysimilar to influenza A virus, there are some differences. For example,influenza B virus does not have a M2 protein with ion channel activity.Instead, the NB protein, a product of the NA gene, likely has ionchannel activity and thus a similar function to the influenza A virus M2protein. Similarly, influenza C virus does not have a M2 protein withion channel activity. However, the CM1 protein is likely to have thisactivity.

Thogotovirus

Thogotoviruses (THOV) represent a new genus in the family ofOrthomyxoviridae. They are transmitted by ticks and have been found indomestic animals, including camels, goats, and cattle. Consequently,THOV can replicate in tick and vertebrate cells. The THOV genomecomprises six segments of single-stranded, negative-sense RNA. Theproteins encoded by the three largest segments show significant homologyto the influenza virus polymerase proteins PB2, PB1, and PA. Segment 5encodes a protein related to influenza virus NP. The THOV glycoprotein,which is encoded by segment 4, is not homologous to either influenzavirus HA or NA, but it shows sequence similarity to the Baculovirusglycoprotein. The smallest segment is thought to encode a matrix proteinand does not resemble any of the influenza virus proteins. Likeinfluenza virus, both the 3′ and 5′ ends of the vRNA are required forpromoter activity, and this activity is located in the terminal 14 and15 nucleotides of the 3′ and 5′ ends of the vRNA, respectively.

The mRNA synthesis of THOV is primed by host cell-derived capstructures. However, in contrast to influenza virus, only the capstructures (without additional nucleotides) are cleaved from cellularmRNAs (Albo et al., 1996; Leahy et al., 1997; Weber et al., 1996). Invitro cleavage assays revealed that both the 5′ and 3′ ends of vRNA arerequired for endonuclease activity (Leahy et al., 1998), but addition ofa model cRNA promoter does not stimulate endonuclease activity (Leahy etal., 1998), as has been shown for influenza virus (Cianci et al., 1995;Hagen et al., 1994). A ‘hook’ structure has been proposed for THOV(Leahy et al., 1997; Weber et al., 1997), which is similar to thecorkscrew structure proposed for influenza virus (Flick et al., 1996).This ‘hook’ structure, however, is only found in the THOV vRNA promoter.The cRNA promoter sequence does not allow the formation of base pairsbetween positions 2 and 9, and between 3 and 8 at the 5′ end of thecRNA. Alterations at positions 3 or 8 to allow base-pairing betweenthese nucleotides stimulates endonuclease activity, which is strongsupporting evidence of the proposed ‘hook’ structure (Leahy et al.,1998). Moreover, this structure might be crucial for the regulation ofthe THOV life cycle; the vRNA promoter, forming the ‘hook’ structure,may stimulate PB2 endonuclease activity, thereby allowing transcription.The cRNA promoter, in contrast, may not form the ‘hook’ structure andmay therefore be unable to stimulate endonuclease activity, thusresulting in replication.

Bunyaviridae

The family Bunyaviridae includes several viruses that cause hemorrhagicor encephalitic fevers in humans (e.g., Rift fever valley, Hantaan, LaCrosse, and Crimean-Congo hemorrhagic fever). The spherical andenveloped virions contain three segments of single-stranded,negative-sense RNA (reviewed in Elliott, 1997). The largest segment (L)encodes the viral RNA polymerase protein (L protein), whereas the Msegment encodes the two viral glycoproteins G1 and G2, and anonstructural protein (NSm). The smallest segment (S) encodes thenucleocapsid protein (N) and a second nonstructural protein (NSs). Virusreplication and transcription take place in the cytoplasm, and newlyassembled virions bud through the membranes of the Golgi apparatus.

Bridgen & Elliott (1996) have established a reverse genetics system togenerate infectious Bunyamwera virus entirely from cloned cDNAs. Theyfollowed a strategy first described by Schnell et al. (1994) for rabiesvirus: intracellular transcription of a cDNA coding for thepositive-sense antigenomic RNA (but not for the negative-sense genomicRNA) in cells expressing the viral polymerase and nucleoprotein. Bridgen& Elliott (1996) infected HeLaT4+ cells with vaccinia virus expressingT7 polymerase and transfected these cells with plasmids expressingproteins encoded by the S, M, and L segments. They then transfectedthese cells with three plasmids encoding full-length anti-genomic cDNAsflanked by the T7 polymerase promoter and the hepatitis delta virusribozyme. To increase the number of bunyavirus particles relative to thenumber of vaccinia virus particles, the authors used mosquito cells inwhich Bunyamwera but not Vaccinia virus replicates. This protocol can beused not only to genetically engineer Bunyaviridae, but also generatereassortant viruses that cannot easily be obtained by coinfecting cellswith different Bunyaviridae strains.

To study bunyavirus promoter elements and the viral proteins that arerequired for transcription and replication, Dunn et al. (1995) clonedthe CAT gene in the negative-sense orientation between the 5′ and 3′nontranslated regions of the Bunyamwera S RNA segment. Cells weretransfected with constructs expressing the proteins encoded by the L andS segment and were then transfected with in vitro transcribed RNA, whichresulted in CAT activity. The bunyavirus S segment encodes two proteins,N and NSs, in overlapping reading frames. To determine whether both ofthese proteins are required for transcription and replication,constructs expressing only N or NSs were tested for CAT activity. Nprotein expression, together with L protein, resulted in CAT activity,whereas no CAT activity was detected with the NSs expression construct.Thus, the L and N proteins are sufficient for transcription andreplication of a bunyavirus-like RNA.

As with influenza virus, the terminal sequences of bunyavirus RNAs arecomplementary and highly conserved. It has therefore been assumed thatthese sequence elements define the bunyaviral promoter and are crucialfor promoter activity. Deletion of five nucleotides at the 3′ end of theviral RNA drastically reduces CAT expression (Dunn et al., 1995). Incontrast, addition of two nucleotides at the 5′ end, or of 11 or 35nucleotides at the 3′ end does not abolish CAT expression (Dunn et al.,1995). Therefore, like the influenza virus polymerase complex, thebunyavirus polymerase protein can apparently start transcription and/orreplication internally.

The invention will be further described by the following non-limitingexamples.

Example 1

Materials and Methods

Cells and viruses. 293T human embryonic kidney cells and Madin-Darbycanine kidney cells (MDCK) were maintained in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal calf serum and in modifiedEagle's medium (MEM) containing 5% newborn calf serum, respectively. Allcells were maintained at 37° C. in 5% CO₂. Influenza viruses A/WSN/33(H1N1) and A/PR/8/34 (H1N1) were propagated in 10-day-old eggs.

Construction of plasmids. To generate RNA polymerase I constructs,cloned cDNAs derived from A/WSN/33 or A/PR/8/34 viral RNA wereintroduced between the promoter and terminator sequences of RNApolymerase I. Briefly, the cloned cDNAs were amplified by PCR withprimers containing BsmBI sites, digested with BsmBI, and cloned into theBsmBI sites of the pHH21 vector which contains the human RNA polymeraseI promoter and the mouse RNA polymerase I terminator, separated by BsmBIsites (FIG. 2). The PB2, PB1, PA, HA, NP, NA, M, and NS genes of theA/WSN/33 strain were PCR-amplified by use of the following plasmids:pSCWPB2, pGW-PB 1, and pSCWPA (all obtained from Dr. Debi Nayak at theUniversity of California Los Angeles), and pWH17, pWNP152, pT3WNA15(Castrucci et al., 1992), pGT3WM, and pWNS1, respectively. The PB1 geneof influenza A/PR/8/34 virus was amplified by using pcDNA774 (PB1)(Perez et al., 1998) as a template. See FIG. 6 for the sequences of theprimers. To ensure that the genes were free of unwanted mutations,PCR-derived fragments were sequences with an autosequencer (AppliedBiosystem Inc., CA, USA) according to the protocol recommended by themanufacturer. The cDNAs encoding the HA, NP, NA, and M1 genes ofA/WSN/33 virus were cloned as described (Huddleston et al., 1982) andsubcloned into the eukaryotic expression vector pCAGGS/MCS (controlledby the chicken β-actin promoter) (Niwa et al., 1991), resulting inpEWSN-HA, pCAGGS-WSN-NP0-14, pCAGGS-WNA15, and pCAGGS-WSN-M1-2/1,respectively. The M2 and NS2 genes from the A/PR/8/34 virus wereamplified by PCR and cloned into pCAGGS/MCS, yielding pEP24c andpCA-NS2. Finally, pcDNA774(PB1), pcDNA762(PB2), and pcDNA787(PA) wereused to express the PB2, PB1, and PA proteins under control of thecytomegalovirus promoter (Perez et al., 1998).

Generation of infectious influenza particles. 293T cells (1×10⁶) weretransfected with a maximum of 17 plasmids in different amounts with useof Trans IT LT-1 (Panvera, Madison, Wis.) according to themanufacturer's instructions. Briefly, DNA and transfection reagent weremixed (2 μl Trans IT-LT-1 per μg of DNA), incubated at room temperaturefor 45 minutes and added to the cells. Six hours later, theDNA-transfection reagent mixture was replaced by Opti-MEM (Gibco/BRL,Gaithersburg, Md.) containing 0.3% bovine serum albumin and 0.01% fetalcalf serum. At different times after transfection, viruses wereharvested from the supernatant and titrated on MDCK cells. Since helpervirus was not required by this procedure, the recovered transfectantviruses were analyzed without plaque purification.

Determination of the percentage of plasmid-transfected cells producingviruses. Twenty-four hours after transfection, 293T cells were dispersedwith 0.02% EDTA into single cells. The cell suspension was then diluted10-fold and transferred to confluent monolayers of MDCK cells in 24-wellplates. Viruses were detected by the hemagglutination assay.

Immunostaining assay. Nine hours after infection with influenza virus,cells were washed twice with phosphate-buffered saline (PBS) and fixedwith 3.7% paraformaldehyde (in PBS) for 20 minutes at room temperature.Next, they were treated with 0.1% Triton X-100 and processed asdescribed by Neumann et al. (1997).

Results

Generation of infectious virus by plasmid-driven expression of viral RNAsegments, three polymerase subunits and NP protein. Althoughtransfection of cells with a mixture of RNPs extracted from purifiedvirions results in infectious influenza particles, this strategy is notlikely to be efficient when used with eight different in vitro generatedRNPs. To produce infectious influenza viruses entirely from cDNAs, eightviral RNPs were generated in vivo. Thus, plasmids were prepared thatcontain cDNAs for the full-length viral RNAs of the A/WSN/33 virus,flanked by the human RNA polymerase I promoter and the mouse RNApolymerase I terminator. In principle, transfection of these eightplasmids into eukaryotic cells should result in the synthesis of alleight influenza vRNAs. The PB2, PB1, PA and NP proteins, generated bycotransfection of protein expression plasmids, should then assemble thevRNAs into functional vRNPs that are replicated and transcribed,ultimately forming infectious influenza viruses (FIG. 3). 1×10⁶ 293Tcells were transfected with protein expression plasmids (1 μg ofpcDNA762(PB2), 1 μg of pcDNA774(PB1), 0.1 μg of pcDNA787(PA), and 1 μgof pCAGGS-WSN-NP0/14) and 1 μg of each of the following RNA polymerase Iplasmids (pPo1I-WSN-PB2, pPo1I-WSN-PB1, pPo1I-WSN-PA, pPo1I-WSN-HA,pPo1I-WSN-NP, pPo1I-WSN-NA, pPo1I-WSN-M, and pPo1I-WSN-NS). The decisionto use a reduced amount of pcDNA787(PA) was based on previousobservations (Mena et al., 1996), and data on the optimal conditions forgeneration of virus-like particles (VLPs) (data not shown). Twenty-fourhours after transfection of 293T cells, 7×10³ pfu of virus per ml wasfound in the supernatant (Experiment 1, Table 1), demonstrating for thefirst time the capacity of reverse genetics to produce influenza A virusentirely from plasmids.

TABLE 1 Plasmid sets used to produce influenza virus from cloned cDNA*Experiment 1 2 3 4 5 6 7 8 RNA polymerase I plasmids for:^(†) PB1 + + −− − − − − PR8-PB1 − − + + + + + + PB2 + + + + + + + + PA + + + + + + + +HA + + + + + + + + NP + + + + + + + + NA + + + + + + + +M + + + + + + + + NS + + + + + + + + Protein expression plasmids for:PB1 + + + + − + + + PB2 + + + + + − + + PA + + + + + + − +NP + + + + + + + − HA − + − + + + + + NA − + − + + + + + M1 − +− + + + + + M2 − + − + + + + + NS2 − + − + + + + + Virus titer 7 × 10³ 7× 10³ 1 × 10³ 3 × 10⁴ 0 0 0 0 (pfu/ml) *293T cells were transfected withthe indicated plasmids. Twenty-four (Experiments 1 and 2) or forty-eighthours (Experiments 3-8) later, the virus titer in the supernatant wasdetermined in MDCK cells. ^(†)Unless otherwise indicated, plasmids wereconstructed with cDNAs representing the RNAs of A/WSN/33 virus.

Efficiency of influenza virus production with coexpression of all viralstructural proteins. Although expression of the viral NP and polymeraseproteins is sufficient for the plasmid-driven generation of influenzaviruses, it was possible that the efficiency could be improved. Inprevious studies, the expression of all influenza virus structuralproteins (PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2) resulted in VLPsthat contained an artificial vRNA encoding a reporterchloramphenicol-acetyltransferase gene (Mena et al., 1996). Thus, theavailability of the entire complement of structural proteins, instead ofonly those required for viral RNA replication and transcription, mightimprove the efficiency of virus production. To this end, 293T cells weretransfected with optimal amounts of viral protein expression plasmids(as judged by VLP production; unpublished data): 1 μg of pcDNA762(PB2)and pcDNA774(PB1); 0.1 μg of pcDNA787(PA); 1 μg of pEWSN-HA,pCAGGS-WSN-NP0/14, and pCAGGS-WNA15; 2 μg of pCAGGS-WSN-M1-2/1; 0.3 μgof pCA-NS2; and 0.03 μg of pEP24c (for M2), together with 1 μg of eachRNA polymerase I plasmid (Experiment 2, Table 1). A second set of cellswas transfected with the same set of RNA polymerase I plasmids, with theexception of the PB1 gene, for which pPo1I-PR/8/34-PB1 was substitutedin an effort to generate a reassortant virus, together with plasmidsexpressing only PA, PB1, PB2, and NP (Experiment 3, Table 1) or thoseexpressing all the influenza structural proteins (Experiment 4, Table1). Yields of WSN virus did not appreciably differ at 24 hours(Experiments 1 and 2, Table 1) or at 36 hours (data not shown)post-transfection. However, more than a 10-fold increase in yields ofthe virus with PR/8/34-PB1 was found when all the influenza viralstructural proteins were provided (Experiments 3 and 4, Table 1).Negative controls, which lacked one of the plasmids for the expressionof PA, PB1, PB2, of NP proteins, did not yield any virus (Experiments5-8, Table 1). Thus, depending on the virus generated, expression of allinfluenza A virus structural proteins appreciably improved theefficiency of the reverse genetics method.

Next, the kinetics of virus production after transfection of cells wasdetermined using the set of plasmids used to generate a virus with theA/PR/8/34-PB1 gene. In two of three experiments, virus was firstdetected at 24 hours after transfection. The titer measured at thattime, >10³ pfu/ml, had increased to >10⁶ pfu/ml by 48 hours aftertransfection (Table 2). To estimate the percentage ofplasmid-transfected cells that were producing viruses, 293T cells weretreated with EDTA (0.02%) at 24 hours after transfection to disperse thecells, and then performed limiting dilution studies. In this experiment,no free virus was found in the culture supernatant at this time point.The results indicated that 1 in 10^(3.3) cells was generating infectiousvirus particles.

TABLE 2 Kinetics of virus production after plasmid transfection into293T cells* Virus titers in culture Hours after supernatant (pfu/ml)plasmid Experiment transfection 1 2 3 6 0 ND ND 12 0 ND 0 18 0 ND 0 24 0  2 × 10³ 6 × 10³ 30 ND   5 × 10⁴ 9 × 10⁴ 36 6 × 10² >1 × 10⁵ 7 × 10⁵ 42ND >1 × 10⁶ 5 × 10⁶ 48 8 × 10⁴ >1 × 10⁶ 1 × 10⁷ *293T cells weretransfected with eight RNA polymerase I plasmids encoding A/WSN/33 virusgenes with the exception of PB1 gene, which is derived from A/PR/8/34virus, and nine protein expression plasmids as described in the text. Atdifferent time points, we titrated virus in the culture supernatant inMDCK cells. ND = not done.

Recovery of influenza virus containing the FLAG epitope in the NAprotein. To verify that the new reverse genetics system allowed theintroduction of mutations into the genome of influenza A viruses, avirus containing a FLAG epitope (Castrucci et al., 1992) in the NAprotein was generated. 293T cells were transfected with an RNApolymerase I plasmid (pPo1I-WSN-NA/FL79) that contained a cDNA encodingboth the NA protein and a FLAG epitope at the bottom of the protein'shead, together with the required RNA polymerase I and protein expressionplasmids. To confirm that the recovered virus (PR8-WSN-FL79) did in factexpress the NA-FLAG protein, immunostaining assays of cells infectedwith PR8-WSN-FL79 or A/WSN/33 wild-type virus was performed. Amonoclonal antibody to the FLAG epitope detected cells infected withPR8-WSN-FL79, but not those infected with wild-type virus (FIG. 4).Recovery of the PR8-WSN-FL79 virus was as efficient as that for theuntagged wild-type virus (data not shown). These results indicate thatthe new reverse genetics system allows one to introduce mutations intothe influenza A virus genome.

Generation of infectious influenza virus containing mutations in the PAgene. To produce viruses possessing mutations in the PA gene, two silentmutations were introduced creating new recognition sequences forrestriction endonucleases (Bsp120I at position 846 and PvuII at position1284 of the mRNA). Previously, it was not possible to modify this geneby reverse genetics, because of the lack of a reliable selection system.Transfectant viruses, PA-T846C and PA-A1284 were recovered. Therecovered transfectant viruses were biologically cloned by twoconsecutive limiting dilutions. To verify that the recovered viruseswere indeed transfectants with mutations in the PA gene, cDNA for the PAgene was obtained by reverse transcriptase-PCR. As shown in FIG. 5,PA-T846C and PA-A1284C viruses had the expected mutations within the PAgene, as demonstrated by the presence of the newly introducedrestriction sites. PCR of the same viral samples and primers without thereverse transcription step failed to produce any products (data notshown), indicating that the PA cDNA was indeed originated from vRNAinstead of the plasmid used to generate the viruses. These resultsillustrate how viruses with mutated genes can be produced and recoveredwithout the use of helper viruses.

Discussion

The reverse genetics systems described herein allows one to efficientlyproduce influenza A viruses entirely from cloned cDNAs. Bridgen andElliott (1996) also used reverse genetics to generate a Bunyamwera virus(Bunyaviridae family), but it contains only three segments ofnegative-sense RNA, and the efficiency of its production was low, 10²pfu/10⁷ cells. Although the virus yields differed among the experiments,consistently >10³ pfu/10⁶ cells was observed for influenza virus, whichcontains eight segments. There are several explanations for the highefficiency of the reverse genetics system described hereinabove. Insteadof producing RNPs in vitro (Luytjes et al., 1989), RNPs were generatedin vivo through intracellular synthesis of vRNAs using RNA polymerase Iand through plasmid-driven expression of the viral polymerase proteinsand NP. Also, the use of 293T cells, which are readily transfected withplasmids (Goto et al., 1997), ensured that a large population of cellsreceived all of the plasmids needed for virus production. In addition,the large number of transcripts produced by RNA-polymerase I, which isamong the most abundantly expressed enzymes in growing cells, likelycontributed to the overall efficiency of the system. These features ledto a correspondingly abundant number of vRNA transcripts and adequateamounts of viral protein for encapsidation of vRNA, formation of RNPs inthe nucleus, and export of these complexes to the cell membrane, wherenew viruses are assembled and released.

Previously established reverse genetics systems (Enami et al., 1990;Neumann et al., 1994; Luytjes et al., 1989; Pleschka et al., 1996)require helper-virus infection and therefore selection methods thatpermit a small number of transfectants to be retrieved from a vastnumber of helper viruses. Such strategies have been employed to generateinfluenza viruses that possess one of the following cDNA-derived genes:PB2 (Subbarao et al., 1993), HA (Enami et al., 1991: Horimoto et al.,1994), NP (Li et al., 1995), NA (Enami et al., 1990), M (Castrucci etal., 1995; Yasuda et al., 1994), and NS (Enami et al., 1991). Most ofthe selection methods, except for those applicable to the HA and NAgenes, rely on growth temperature, host range restriction, or drugsensitivity, thus limiting the utility of reverse genetics forfunctional analysis of the gene products. Even with the HA and NA genes,for which reliable antibody-driven selection systems are available, itis difficult to produce viruses with prominent growth defects. Incontrast, the reverse genetics system described herein does not requirehelper virus and permits one to generate transfectants with mutations inany gene segment or with severe growth defects. This advantage isdemonstrated in FIG. 5, which the recovery of transfectant viruses witha mutated PA gene. Having the technology to introduce any viablemutation into the influenza A virus genome will enable investigators toaddress a number of long-standing issues, such as the nature ofregulatory sequences in nontranslated regions of the viral genome,structure-function relationships of viral proteins, and the molecularbasis of host-range restriction and viral pathogenicity.

Although inactivated influenza vaccines are available, their efficacy issuboptimal due partly to their limited ability to elicit local IgA andcytotoxic T cell responses. Clinical trials of cold-adapted liveinfluenza vaccines now underway suggest that such vaccines are optimallyattenuated, so that they will not cause influenza symptoms, but willstill induce protective immunity (reviewed in Keitel & Piedra, 1998).However, preliminary results indicate that these live virus vaccineswill not be significantly more effective than the best inactivatedvaccine (reviewed in Keitel. & Piedra, 1998), leaving room for furtherimprovement. One possibility would be to modify a cold-adapted vaccinewith the reverse genetics system described above. Alternatively, onecould start from scratch by using reverse genetics to produce a “master”influenza A strain with multiple attenuating mutations in the genes thatencode internal proteins. The most intriguing application of the reversegenetics system described herein may lie in the rapid production ofattenuated live-virus vaccines in cases of suspected pandemics involvingnew HA or NA subtypes of influenza virus.

This new reverse genetics system will likely enhance the use ofinfluenza viruses as vaccine vectors. The viruses can be engineered toexpress foreign proteins or immunogenic epitopes in addition to theinfluenza viral proteins. One could, for example, generate viruses withforeign proteins as a ninth segment (Enami et al., 1991) and use them aslive vaccines. Not only do influenza viruses stimulate strongcell-mediated and humoral immune responses, but they also afford a widearray of virion surface HA and NA proteins (e.g., 15 HA and 9 NAsubtypes and their epidemic variants), allowing repeated immunization ofthe same target population.

Influenza VLPs possessing an artificial vRNA encoding a reporter genehave been produced by expressing viral structural proteins and vRNA withthe vaccinia-T7 polymerase system (Mena et al., 1996). Using reversegenetics, one can now generate VLPs containing vRNAs that encodeproteins required for vRNA transcription and replication (i.e., PA, PB1,PB2, and NP), as well as vRNAs encoding proteins of interest. Such VLPscould be useful gene delivery vehicles. Importantly, their lack of genesencoding viral structural proteins would ensure that infectious viruseswill not be produced after VLP-gene therapy. Since the influenza virusgenome is not integrated into host chromosome, the VLP system would besuitable for gene therapy in situations requiring only short-termtransduction of cells (e.g., for cancer treatment). In contrast toadenovirus vectors (Kovesdi et al., 1997), influenza VLPs could containboth HA and NA variants, allowing repeated treatment of targetpopulations.

The family Orthomyxoviridae comprises influenza A, B, and C viruses, aswell as the recently classified Thogotovirus. The strategy forgenerating infectious influenza A viruses entirely from cloned cDNAsdescribed herein would apply to any orthomyxovirus, and perhaps to othersegmented negative-sense RNA viruses as well (e.g., Bunyaviridae,Arenaviridae). The ability to manipulate the viral genome withouttechnical limitations has profound implications for the study of virallife cycles and their regulation, the function of viral proteins and themolecular mechanisms of viral pathogenicity.

Example 2

Materials and Methods

Cells and viruses. 293T human embryonic kidney cells and Madin-Darbycanine kidney cells (MDCK) were maintained in DMEM supplemented with 10%FCS and in MEM containing 5% newborn calf serum, respectively. The 293Tcell line is a derivative of the 293 line, into which the gene for thesimian virus 40 T antigen was inserted (DuBridge et al., 1987). Allcells were maintained at 37° C. in 5% CO₂. Influenza virusA/Udorn/307/72 (H3N2) (Udorn) was propagated in 10-day-old eggs.

Construction of plasmids. The cDNA of Undorn virus was synthesized byreverse transcription of viral RNA with an oligonucleotide complementaryto the conserved 3′ end of viral RNA, as described by Katz et al.(1990). The cDNA was amplified by PCR with M gene-specificoligonucleotide primers containing BsmBI sites, and PCR products werecloned into the pT7Blueblunt vector (Novagen, Madison, Wis.). Theresulting construct was designated pTPo1UdM. After digestion with BsmBI,the fragment was cloned into the BsmBI sites of the pHH21 vector, whichcontains the human RNA polymerase I promoter and the mouse RNApolymerase I terminator, separated by BsmBI sites (Neumann et al.,1999), resulting in pPo1IUdM. Plasmids derived from pHH21 for theexpression of vRNA are referred to as “Po1I” constructs in this report.

The M mutants were constructed as follows. pTPo1IUdM was first amplifiedby inverse PCR (Ochman et al., 1988) using the back-to-back primersM2104R (5′-AAGAGG GTCACTTGAATCG-3′; SEQ ID NO:1) and M2V27T(5′-ACTGTTGCTGCGAGTATC-3′; SEQ ID NO:2) and M2A30P(5′-GTTGTTGCTCCAACTATC-3′; SEQ ID NO:3) and M2S31N(5′-GTTGTTGCTGCGAACATC-3′; SEQ ID NO:4) and M2del29-31(5′-GTTGTTATCATTGGGATCTTGC-3′; SEQ ID NO:5), and the back-to-backprimers M2128R (5′-CCCAATGATACTCGCAGC-3′; SEQ ID NO:6) and M2W41A(5′-ATCTTGCACTTGATATTGGCAATTC-3′; SEQ ID NO:7), and the back-to-backprimers M2HATMR(5′-CACCAGTGAACTGGCGACAGTTGAGTAGATCGCCAGAATGTCACTTGAATCGTTGCATCTGC-3′; SEQ ID NO:8) and M2HATM(5′-CTTTTGGTCTCCCTGGGGGCAATCAGTTTCTGGATGGATCGTCTTTTTTTC AAATGC-3′; SEQID NO:9), and M2NATMR(5′-GCTTAGTATCAATTGTATTCCATTTATGATTGATATCCAAATGCTGTCACTTGAATCGTTGCATCTGC-3′SEQ ID NO:10) and M2NATM(5′-ATTATAGGAGTCGTAATGTGTATCTCAGGGATTACCATAATAGATCGTCT TTTTTTCAAATGC-3′;SEQ ID NO: 11).

The PCR products were phosphorylated, self-ligated, and propagated in E.coli strain DH5α, and then digested with BsmBI and cloned into the BsmBIsites of the pHH21 vector. The resulting constructs were designatedpPo1M2V27T, pPo1IM2A30P, pPo1IM2S31N, pPo1IM2del29-31, pPo1IM2W41A,pPo1IM2HATM, and pPo1IM2NATM. All of the constructs were sequenced toensure that unwanted mutations were not present. The plasmids for theexpression of the HA (pEWSN-HA), NP (pCAGGS-WSN-NP0/14), NA(pCAGGS-WNA15), M1 (pCAGGS-WSN-M1-2/1) proteins of A/WSN/33 (H1N1)virus, and the M2 (pEP24c), NS2 (pCANS2), PB1 (pcDNA774), PB2(pcDNA762), and PA (pcDNA787) of A/Puerto Rico/8/34 (H1N1) virus aredescribed in Neumann et al. (1999).

Plasmid-driven Reverse Genetics. Transfectant viruses were generated asreported in Neumann et al. (1999). Briefly, 17 plasmids (8 Po1Iconstructs for 8 RNA segments and 9 protein-expression constructs for 9structural proteins) were mixed with transfection reagent (2 μL of TransIT LT-1 (Panvera, Madison, Wis.) per μg of DNA), incubated at roomtemperature for 15 minutes, and added to 1×10⁶ 293T cells. Six hourslater, the DNA-transfection reagent mixture was replaced by Opti-MEM(GIBCO/BRL) containing 0.3% BSA and 0.01% FCS. Forty-eight hours later,viruses in the supernatant were plaque-purified in MDCK cells once andthen inoculated into MDCK cells for the production of stock virus. The Mgenes of transfectant viruses were sequenced to confirm the origin ofthe gene and the presence of the intended mutations and to ensure thatno unwanted mutations were present. In all experiments, the transfectionviruses contained only the M gene from Undorn virus and the remaininggenes from A/WSN/33.

Replicative properties of the transfectant viruses. MDCK cells induplicate wells of 24-wells plates were infected with wild-type andmutant viruses at a multiplicity of infection (MOI) of 0.001plaque-forming units (PFU) per cell, overlaid with MEM medium containing0.5 μg of trypsin per ml, and incubated at 37° C. At different times,supernatants were assayed for infectious virus in plaque assays on MDCKcells.

To investigate the amantadine sensitivity of mutant viruses, the viruseswere titrated in MDCK cells in the presence of different concentrationsof the drug.

M2 incorporation into viruses. Transfectant viruses were grown in MDCKcells containing 0.5 μg of trypsin per ml. Viruses were purified throughsix-step sucrose gradients (20, 30, 35, 40, 45, and 50%) for 2.5 hoursat 50,000 g at 4° C. Virus was resuspended in PBS and stored in aliquotsat −80° C. Purified virus was resuspended in the lysis buffer (0.6 MKCl, 50 mM Tris-Cl [pH 7.5], 0.5% Triton X-100). The viral lysates wereplaced on 15% SDS-polyacrylamide gels, which then wereelectrotransferred to polyvinylidene difluoride (PVDF) membrane. Themembrane was blocked overnight at 4° C. with 5% skimmed milk in PBS, andthen incubated with the 14C2 anti-M2 monoclonal antibody (kindlyprovided by Dr. R. Lamb) and anti-WSN-NP monoclonal antibody for 1 hourat room temperature. The membrane was washed three times with PBScontaining 0.05% Tween-20. Bound antibodies were detected with aVECTASTAIN ABC kit (Vector) and the Western immunoblot ECL system(Amersham). Signal intensities were quantified with an Alpha Imager 2000(Alpha Innotech Corporation).

Experimental infection. Five-week-old female BALB/c mice, anesthetizedwith isoflurane, were infected intranasally with 50 μl (5.0×10³ PFU) ofvirus. Virus titers in organs were determined 3 days after infectionwith MDCK cells, as described (Bilsel et al., 1993).

Results

Generation of influenza A viruses containing mutations in the M2protein. The TM domain of the M2 protein is modeled to have an a helicalstructure (Duff et al., 1992; Sugrue and Hay, 1991; Sansom and Kerr,1993). Mutations at residues V-27, A-30, S-31, G-34, and L-38, all ofwhich are located on the same face of the α helix, alter the propertiesof the M2 ion channel (Grambas et al., 1992; Pinto et al., 1992; Wang etal., 1993). To determine whether the ion channel activity of M2 isessential for viral replication, five plasmids were constructed and usedto generate mutant viruses possessing changes in the M2 TM domain (FIG.7). The whole-cell currents of the mutant proteins expressed in oocytesof Xenopus laevis, were measured by Holsinger et al. (1994), using atwo-electrode voltage-clamp procedure. None of three mutants, i.e.,M2A30P, M2W41A, and M2del29-31, had functional ion channel activity ateither neutral or low pH. M2V27T and M2S31N, which showed ion channelactivity at low pH (Holsinger et al., 1994), were used as positivecontrols.

To generate mutant viruses by plasmid-driven reverse genetics (Neumannet al., 1999), 293T cells were transfected with nine protein-expressionplasmids and eight others for the production of viral RNA segments thatencoded all A/WSN/33 (H1N1) viral genes except the M gene, which wasderived from the A/Udorn/307/72 (H3N2) (Undorn) virus (wild-type). Thecorresponding transfectant viruses were designated M2V27T, M2A30P,M2S31N, M2W41A, M2del29-31, and WSN-UdM, for the virus containing theparental Undorn M gene.

To determine the efficiency of virus generation, viruses were titratedin the culture supernatant of 293T cells at 48 hours post-transfectionusing MDCK cells. As shown in Table 3, more than 10⁵ transfectantviruses with the wild-type or mutant M gene were present. Thus, allviruses bearing M2 mutations and the virus possessing the wild-typeUdorn M gene were generated with similar efficiency. The transfectantviruses were plaque-purified once in MDCK cells and then inoculated intoMDCK cells to make virus stocks. The stability of the introducedmutations was analyzed by sequencing the M gene segments of thetransfectant viruses after ten passages in MDCK cells. No revertantswere found.

TABLE 3 Virus titers in the supernatant of 293T cells after plasmidtransfection^(a) Virus Titers (PFU/ml) Wild type 1.9 × 10⁵ M2V27T 6.0 ×10⁵ M2A30P 1.1 × 10⁵ M2S31N 1.2 × 10⁶ M2W41A 1.2 × 10⁶ M2del29-31 1.7 ×10⁶ M2HATM 2.2 × 10⁴ M2NATM 2.2 × 10³ ^(a)293T cells were transfectedwith eight plasmids for the production of A/WSN/33 vRNA (excluding the Mgene, which was derived form A/Udorn/72 virus) and nine proteinexpression plasmids, as described in Materials and Methods. At 48 hoursposttransfection, virus in the supernatant of 293T cell cultures wastitrated using MDCK cells.

Growth properties of M2 mutant viruses in tissue culture. Next, thegrowth properties of M2 ion channel mutants and wild-type WSN-UdM virusin MDCK cells were compared (FIG. 8). Cells were infected at an MOI of0.001, and yields of virus in the culture supernatant were determined atdifferent times postinfection. The mutant viruses did not differappreciably from the wild-type WSN-UdM virus in either growth rate orthe size of plaques formed at 48 hours (1.5 mm in diameter in 3 days).

To assess the amantadine sensitivity of these viruses, the M2 mutantsand wild-type WSN-UdM viruses were plaqued in MDCK cells in the presenceof different concentrations of amantadine. In cell culture, amantadineproduces two discrete concentration-dependent inhibitory actions againstviral replication. A nonspecific action at concentrations >50 μM,resulting from an increase in the pH of endosomes, inhibits activationof HA membrane fusion activity involved in endocytosis (Daniels et al.,1985); whereas at lower concentrations, 0.1-5 μM, the drug selectivityinhibits viral replication (Appleyard, 1977). As shown in FIG. 9,amantadine markedly reduced the yield of wild-type WSN-UdM virus, aswell as the size of plaques, at each of the three test concentrations.By contrast, at 5 μM of amantadine, the replication of M2 mutant viruseswas either not affected or inhibited only slightly. Substantialinhibition, due to the drug's nonspecific activity, was seen at 50 μM.Thus, all of the M2 mutants were more resistant to amantadine than thewild-type virus.

Generation of transfectant viruses in which the M2 TM domain wasreplaced with that from the HA or NA. Although the M2A30P, M2W41A, andM2del29-31 mutants do not have functional ion channel activity, asassayed by a two-electrode voltage-clamp procedure (Holsinger et al.,1994), they all replicated as well as the wild-type virus in MDCK cells(FIG. 8). Thus, M2 ion channel activity may not be essential for virusreplication, although it could not be ruled out that low-level ionchannel activity was below the sensitivity of the assay.

To determine whether M2 channel ion activity is not essential for viralreplication, chimeric mutant viruses were generated in which the M2 TMdomain was replaced with that from the HA or NA of the A/WSN/33 virus(FIG. 10). When the supernatant of 293T cells which had been transfectedwith plasmids was assayed for virus production, the chimeric mutants(M2HATM and M2NATM) were each viable, but their titers were more thanone log lower than the wild-type WSN-UdM titer (Table 3). The mutantsalso produced pinpoint plaques after 48 hours of growth. Thus, the M2 TMdomain is dispensable for viral replication in vitro.

Growth properties of the M2HATM mutant in tissue culture. Because thetiters of the M2NATM virus stock did not exceed 10⁴ PFU/ml, the M2HATMvirus was employed for further analysis, first by examining the timecourse of progeny virus production by M2HATM versus wild-type WSN-UdMviruses in MDCK cells (FIG. 8). Although M2HATM produced a lower titerthan did the wild-type WSN-UdM virus at 12 and 24 hours postinfection,its maximum titer at 36 hours was almost the same as that of thewild-type virus. This result indicates that the absence of the M2 TMdomain does not drastically impair the replicative ability of the virusin tissue culture.

Incorporation of mutant M2 molecules into virions. Conceivably, the M2point and chimeric mutants possessed some residual ion channel activity,so that increased incorporation of the M2 protein into virions couldcompensate for any defect in this function. Therefore, the efficiency ofincorporation of the wild-type and mutant M2s into influenza virions wascompared using Western blot analysis after standardization based on theintensity of NP (FIG. 11). Virion incorporations of two mutant M2proteins (M2del29-31 and M2HATM) was slightly less than that of thewild-type protein, although the W41A mutant was incorporated moreefficiency. The band detected slightly below the M2 protein in thewild-type is probably a proteolytically cleaved form of M2, as reportedby others (Zebedee and Lamb, 1988). An additional band below the NPprotein that was reactive with anti-NP, but not anti-M2 antibody, is acleavage product of NP (Zhirnov and Bukrinskaya, 1984). Together, theseresults demonstrate that increased incorporation of M2 protein intovirions does not seem to compensate for defective M2 ion channelactivity.

Replication of M2 mutant viruses in mice. To determine the role of M2ion channel activity in vivo, mice were infected with each of the sixmutant viruses (Table 4), which replicated in the lungs as well as ormore efficiently than the wild-type WSN-UdM virus, although the titer ofthe M2del29-31 virus was a log lower than that of the wild-type virus.By contrast, the mutants showed different replicative potentials innasal, turbinates, with neither the M2A30P nor M2del29-31 virusrecovered from such samples in any of the infected mice. M2HATM viruswas not recovered from either the lungs or the nasal turbinates. Theseresults indicate that M2 ion channel activity is necessary for efficientviral replication in vivo. Further, the serum of the infected mice haveantibodies which bind to the immunizing virus (see Example 3).

TABLE 4 Replication of M2 mutants in mice^(a) Mean titers (log₁₀PFU/g) ±SD Virus Nasal turbinate Lung Wild-type 3.9 ± 0.5 6.8 ± 0.3 M2V27T 4.3 ±0.7 7.3 ± 0.3 M2A30P NR^(b) 6.8 ± 0.1 M2S31N 4.3 ± 0.4 7.0 ± 0.2 M2W41A 3.1 ± 2.2^(c) 6.7 ± 0.2 M2del29-31 NR 5.6 ± 0.1 M2HATM NR NR^(a)Five-week-old female BALB/c mice (n = 4), anesthetized withisoflurane, were infected intranasallly with 50 μ1 of virus (5 × 10³PFU). Virus titers in organs were determined 3 days after infection withMDCK cells. ^(b)NR, virus was not recovered from any of the infectedmice (less than 10² PFU/g). ^(c)Virus was recovered from only three ofthe four mice infected.Discussion

A reverse-genetics system (Neumann et al., 1999) was used to generatetransfectant influenza A viruses with changes in the M2 protein TMdomain that are known to block ion channel activity. Despite thisfunctional defect, all of the mutant viruses replicated as efficientlyas the wild-type WSN-UdM virus in vitro. The dispensability of M2 ionchannel activity in viral replication was reinforced by experiments inwhich the TM domain of the M2 protein was replaced with that from the HAor NA. Thus, in in vitro studies, influenza A viruses did not require M2ion channel activity for efficient replication.

M2 ion channel activity is believed to function at an early stage in theviral life cycle, between the steps of host cell penetration anduncoating of viral RNA. Zhirnov et al. (1990) reported that low pHinduces the dissociation of M1 protein from viral RNPs in vitro. Thisobservation lead others to suggest that the introduction of protons intothe interior of virions through M2 ion channel activity in the endosomesis responsible for M1 dissociation from RNP (reviewed by Helenius,1992). If so, how could this process occur in the absence of M2 ionchannel activity or the M2 TM domain? Immunoelectron microscopy of theHA protein in virosomes exposed to low pH demonstrated that, in theabsence of target membranes, the N-terminal fusion peptide of the HA2subunit was inserted into the same membrane site where HA was anchored(Wharton et al., 1995). Therefore, one possibility is that the fusionpeptide of the HA maybe inserted into the viral envelope, forming poresin the viral membrane that permit the flow of protons from the endosomeinto virus interior, resulting in disruption of RNP-M1 interaction.Alternatively, M1 may be able to dissociate from RNP by an entirelydifferent mechanism, including ion channel activity by the TM regions ofother viral membrane proteins, such as the HA, the NA or both.

What is the origin of the M2 ion channel? The M2 ion channel activitywas originally discovered with A/fowl plague/Rostock/34 (FPV Rostock)strain, which has intracellularly cleavable HA (Sugreu et al., 1990;Ohuchi et al., 1994; Takeuchi and Lamb, 1994). In this strain, the HAundergoes a low pH-induced conformational change in the trans-Golginetwork in the absence of M2 ion channel activity, which raises the pHin this compartment. Hence, in the past, influenza A viruses may havebeen equipped with an M2 protein that promoted an increase in the pH ofthe trans-Golgi network, to a level above which conformational changesoccur in the intracellularly cleavable HA. As influenza A viruseswithout intracellularly cleavable HAs began to appear, there was lessselective pressure to maintain high ion channel activity associated withthe M2 protein. Consequent decreases in this activity may have beensufficient to allow dissociation of M1 from RNP. Indeed, ion channelactivity differs markedly among the M2 proteins of currently recognizedviruses: for example, fivefold more M2 protein from human Udorn virus(containing intracellularly uncleavable HA) is needed to produce thesame ion channel activity displayed by an equivalent amount of M2 fromFPV Rostock virus (containing intracellularly cleavable HA) (Takeuchiand Lamb, 1994). Conversely, the HAs of some influenza A viruses havechanged from intracellularly uncleavable to cleavable during replicationin chickens (Kawaoka et al., 1984; Horimoto and Kawaoka, 1995; Horimotoet al., 1995), suggesting that M2 protein with limited ion channelactivity could acquire greater activity once a switch to intracellularlycleavable HA has occurred.

The M2 ion channel knock-out and M2HATM viruses replicated reasonablywell in tissue culture, but were highly attenuated in mice, raising thepossibility for their use as live vaccines. Cold-adapted live vaccines,now in clinical trials (reviewed by Maasab and Bryant, 1999), holdconsiderable promise for use in the general population (Sears et al.,1988; Steinhoff et al., 1991; Steinhoff et al., 1990). The major concernis that the limited number of attenuating mutations in such vaccines(Cox et al., 1988; Herlocher et al., 1993) could permit the generationof revertant viruses. Abolishing the M2 ion channel activity, forexample, by replacing the M2 TM domain with that from the HA, wouldgreatly reduce the likelihood of the emergence of revertant viruses.Thus, using our new reverse-genetics system, the generation of influenzaviruses with modified viral genes could lead to the production of safelive influenza vaccines.

To date, four viral proteins have been reported to act as ion channels:M2 of influenza A virus, NB or influenza B virus, and Vpu and Vpr ofhuman immunodeficiency virus type 1 (HIV-1) (Ewart et al., 1996; Pilleret al., 1996; Pinto et al., 1992; Schubert et al., 1996; Sunstrom etal., 1996). Since the replication strategies of influenza type A and Bviruses are very similar, the NB ion channel activity is also thought toplay a role at an early stage of the viral life cycle, although NB stilllacks a demonstrated function in viral replication. Although the Vpuprotein of HIV-1 enhances the release of virus particles from cells(Schubert et al., 1995; Strebel et al., 1988; Terwilliger et al., 1989),its gene can be deleted without completely abrogating HIV-1 replicationin vitro (Cohen et al., 1988; Klimkait et al., 1990; Strebel et al.,1988, 1989). Vpr, another HIV-1 auxiliary protein, is likewise notessential for replication in tissue culture (Dedera et al., 1989).Finally, here, we have shown that M2 ion channel activity is notessential for the life cycle of influenza A viruses. Therefore, ionchannel activities of viral proteins may be an auxiliary function ingeneral, although they can promote more efficient viral replicationunder certain conditions such as in vivo, as shown hereinabove.

Example 3

Materials and Methods

Cells and viruses. 293T human embryonic kidney cells and Madin-Darbycanine kidney (MDCK) cells were maintained in DMEM supplemented with 10%FCS and in MEM containing 5% newborn calf serum, respectively. The 293Tcell line is a derivative of the 293 line, into which the gene for thesimian virus 40 T antigen was inserted (Dubridge et al., 1987). Allcells were maintained at 37° C. in 5% CO₂. M2del29-31 and WSN-UdM(wild-type) viruses were propagated in MDCK cells. A/WSN/33 (H1N1) viruswas propagated in 10-day-old embryonated chicken eggs.

Immunization and protection tests. BALB/c mice (4-week-old female) wereintranasally immunized with 50 μl of 1.1×10⁵ PFU per ml of M2del29-31 orwild-type WSN-UdM viruses. On the second week, four mice were sacrificedto obtain sera, trachea-lung washes, and nasal washes. Two weeks and oneor three months after the vaccination, immunized mice were challengedintranasally, under anesthesia, with 100 LD₅₀ doses of the wild-type WSNvirus. For determination of virus titers, lungs were harvested at day 3and were homogenized and titrated on MDCK cells. The remaining animalswere observed for clinical signs and symptoms of infection for 14 daysafter challenge.

Detection of virus-specific antibody. Serum samples were examined forantibody by ELISA. In this assay, the wells were coated with purifiedWSN virus after treatment with 0.05 M Tris-HCl (pH 7.8) containing 0.5%Triton X-100 and 0.6 M KCl at room temperature and diluted in PBS. Afterincubation of virus-coated plates with test serum samples, boundantibody was detected with rabbit anti-mouse IgA (Kirkegaard & PerryLaboratories Inc., Gaithersburg, Md.) and goat anti-mouse IgG(Boehringer Mannheim, Germany) conjugated to horseradish peroxidase.

Results

In the second week after immunization, virus-specific IgG and IgA wasfound in nasal washes, lung washes and sera of immunized mouse. Notably,virus specific IgG was found in greater levels in mice immunized withM2del29-31 virus in all three sample types, and virus-specific IgA wasfound in lung washes from M2del29-31-immunized mice but was undetectablein lung washes from WSN-UdM-immunized mice (FIG. 12).

The mice were challenged with wild-type virus two weeks, one month ortwo months after immunization and body weights determined for up to 2weeks (FIG. 13). The body weights of mice immunized with M2del29-31virus and challenged with wild-type virus remained relatively constantregardless of the timing between immunization and challenge while thebody weights of mice immunized with wild-type virus and later challengedwith wild-type virus dropped precipitously after challenge regardless ofthe timing between immunization and challenge.

The lungs from some of the mice were harvested at day 3 after challengeto determine virus titers (Table 4). Only mice that were immunized withwild-type virus and challenged with wild-type virus had detectable virusin the lungs. The lack of the presence of virus in lung of immunizedmice which were challenged correlated with survival after challenge.

TABLE 5 Protection against virus challenge in immunized mice^(a) Virustiter in lungs Immunogen No. survivors/no. tested [1og₁₀(PFU/g)] 2 weekscontrol 0/4 7.5 ± 0.1 del29-31 4/4 0 1 month control 0/4 7.4 ± 0.1del29-31 4/4 0 3 months control 0/4 7.2 ± 0.1 del29-31 4/4 0 ^(a)BALB/cmice (4-week-old female) were intranasally immunized with 50 μ1 of 1.1 ×10⁵ PFU per ml of M2del29-31 or wild-type WSN-UdM virus. Two weeks, orone or three months after the vaccination, immunized mice werechallenged intranasally with 100 LD₅₀ doses of the wild-type WSN virus.For determination of virus titers, lungs were harvested at day 3 andwere homogenized and titrated on MDCK cells. The remaining animals wereobserved for clinical signs and symptoms of infection for 14 days afterchallenge.

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1. A method of preparing an attenuated recombinant influenza virus comprising a mutant M2 protein gene, comprising: (i) transfecting an isolated a host cell with a plurality of influenza virus vRNA and influenza virus protein encoding vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) influenza virus vRNA vectors comprising a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the M cDNA comprises a mutation in the transmembrane domain of M2 protein DNA, wherein the mutation is a deletion of residues that include residues between residues 29 to 31, and wherein the mutation is associated with attenuation of the virus in vivo; and b) influenza virus protein encoding vectors comprising a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an influenza A virus ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
 2. The method of claim 1 wherein the host cell is further transfected with a vector encoding a heterologous immunogenic protein of a pathogen.
 3. A method of preparing a recombinant influenza virus comprising a mutant M2 protein gene for a M2 protein which lacks or has reduced activity relative to the corresponding wild-type M2 protein, comprising: (i) transfecting an isolated host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the M cDNA comprises a mutation in the transmembrane domain of M2 protein DNA, wherein the mutation is the deletion of residues 29 to 31 of the transmembrane domain of M2 or is a deletion of one or more of residues 29 to 31 in the transmembrane domain of M2, and wherein the mutation does not substantially alter the in vitro replication of the virus in the absence of amantadine but is associated with attenuation of the virus in vivo; and b) a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
 4. The method of claim 1 wherein the mutation is the deletion of at least residues 29 to
 31. 5. A method of preparing an attenuated recombinant influenza virus comprising a mutant M2 protein gene for a mutant M2 protein which lacks or has reduced activity relative to the corresponding wild-type M2 protein, comprising: (i) transfecting an isolated host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the M cDNA comprises in the transmembrane domain of the M2 protein an alanine at residue 41 or a threonine at residue 27, and wherein the alanine or threonine do not substantially alter the in vitro replication of the virus in the absence of amantadine but is associated with attenuation of the virus in vivo; and b) a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
 6. The method of claim 1 wherein the sequence which includes the mutation comprises to GTTGTTATCATTGGGATCTTGC (SEQ ID NO:5).
 7. A method of preparing an attenuated recombinant influenza virus comprising a mutant M2 protein gene, comprising: (i) transfecting an isolated host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the M cDNA comprises in the transmembrane domain of the M2 protein: a threonine at residue 27, an alanine at residue 41, a deletion of residues that include residues 29 to 31 or a deletion of one or more of residues 29 to 31, and wherein the threonine, alanine or deletion is associated with attenuation of the virus in vivo; and b) a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and (ii) isolating the virus.
 8. The method of claim 7 wherein the deletion is of residues 29 to 31 of the transmembrane domain of M2.
 9. The method of claim 7 wherein the deletion is of at least residues 29 to
 31. 10. The method of claim 7 wherein the amino acid at residue 41 of the M2 protein is an alanine.
 11. The method of claim 7 wherein the amino acid at residue 27 of the M2 protein is a threonine.
 12. The method of claim 7 wherein the deletion is of one or more of residues 29 to 31 in the transmembrane domain of M2.
 13. The method of claim 7 wherein the sequence which includes the deletion comprises GTTGTTATCATTGGGATCTTGC (SEQ ID NO:5). 